Journal: bioRxiv

Article Title: Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines

doi: 10.1101/2024.07.05.602305

Figure Lengend Snippet: A) Mice were immunized with 0.25 μg commercially available bivalent SARS-CoV-2 mRNA vaccine (0.125 μg each variant) in PBS bolus or PNP-0.5-5 hydrogel with or without 1 μg 3M-052 or 20 μg MPLAs at week 0 and boosted with a homologous boost at week eight. Serum collected at designated time points was analyzed for anti-spike (Wuhan-Hu-1) antibodies via ELISA, as well as for other viral variant anti-spike antibodies, IgG subtypes, and neutralizing capacity. A separate cohort of animals primed and boosted in the same fashion were euthanized two weeks post-boost and spleens harvested for ELISpot evaluation of spike specific (WH1) T cells. Anti-WH1 spike IgG endpoint titers for all animals at B) week eight, C) week 26, and D) over time. E) Area under the curve of antibody titer from week 0 to 26 per animal. F) Decay half-life of antibody titer post-boost derived from parametric bootstrapping of titers following each treatment group’s post-boost peak. Data shown as mean ± SEM, n = 1000 simulations. G) Percent infectivity of BA.4/.5 pseudotyped lentivirus at week 13 post-prime and 1:50 serum dilution. H) Spike specific (WH1) IFN-γ producing splenocytes, as a proxy for T cells, at week two post-boost. Data shown as mean ± SEM, n = 4 - 6. Statistical values shown are p values obtained from GLM fitting and Tukey HSD multiple comparison test in JMP. In D, comparisons shown are to bolus control.

Article Snippet: Tetramers were prepared on ice by adding AF647- or PE-Streptavidin (Thermo Scientific, S32357 or BD, 554061) to 6 μM biotinylated wildtype SARS-CoV-2 spike trimer (Sino Biological, 40589-V27B-B) in five steps, once every 20-60 min, for a final molar ratio of 4.1:1 spike protein to dye and concentration of 0.5 μM.

Techniques: Variant Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Derivative Assay, Infection, Comparison, Control

Journal: bioRxiv

Article Title: Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines

doi: 10.1101/2024.07.05.602305

Figure Lengend Snippet: A) Mice were immunized with bivalent SARS-CoV-2 mRNA vaccine in PBS bolus or PNP-0.5-5 hydrogel with or without adjuvants and lymph nodes harvested at weeks 1, 2, and 4. Week one representative flow plots and quantification of B) activated B cells (B220 + MHCII + CD86 + ) and C) light and dark zone B GC cells (B220 + CD95 - GL7 + CD38 - CD86 + CXCR4 - and CD86 - CXCR4 + ). D) Representative flow plots of B GC cells (B220 + CD95 - GL7 + ), E) spike-specific B GC cells (CD38 - Spike ++ ), and F) T follicular helper cells (T FH , CD19 - CD3 + CD4 + CXCR5 + PD-1 + ). Percentages of parent population except Spike ++ B GC cells, which are of grandparent (see supplemental gating strategy). G) Quantification of B GC cell percentage of all B cells. H) Counts of Spike ++ B GC cells. I) Quantification of T FH cell percentage of all CD4 + T cells. J) Ratio of B GC cells to T FH cells as a metric of T FH cell help quality. All data shown as mean ± SEM, n = 5 - 10. Statistical values shown are p values obtained from GLM fitting and Tukey HSD multiple comparison test in JMP.

Article Snippet: Tetramers were prepared on ice by adding AF647- or PE-Streptavidin (Thermo Scientific, S32357 or BD, 554061) to 6 μM biotinylated wildtype SARS-CoV-2 spike trimer (Sino Biological, 40589-V27B-B) in five steps, once every 20-60 min, for a final molar ratio of 4.1:1 spike protein to dye and concentration of 0.5 μM.

Techniques: Comparison